Use of the Yolk Sac to Decipher the Molecular Requirements for Liver Bud Development

 描述(申请人提供)：这项提案的目标是测试一种新的系统，旨在阐明支持从最终或胚胎内胚层(DE)出现早期肝芽的分子。我们建议通过使用卵黄囊来获得这样的见解，它不仅在功能和结构上与DE相似，而且与DE具有几乎相同的转录谱，并且几乎所有早期的肝芽标记都在卵黄囊中表达。尽管已经确定了一些基本的肝芽因子，但这一领域的空白是 了解这些因素中的任何一个的缺失如何改变肝脏萌发的完整转录图谱。最初阻碍肝脏发芽基因功能鉴定的一个问题是，卵黄囊内脏内胚层(VE)早期需要许多肝芽必需基因。卵黄囊是胚外组织，起到原始肠道的作用；促进营养吸收、代谢废物和气体交换。许多早期的肝脏标志物在卵黄囊中表达，遗传网络的保守性支持一种假设，即在哺乳动物的进化过程中，卵黄囊参与了现有的DE遗传程序。虽然可诱导的Cre策略现在可以绕过VE中的遗传要求，但解剖/分离足够数量的早期肝芽组织的困难阻碍了对正常组织和突变组织的深入全基因组分析。为了克服这些困难，我们建议使用卵黄囊作为早期肝芽的替代品，并对突变的和WT卵黄囊进行转录组分析，以产生在肝芽中起类似作用的候选因子。这一策略将在缺乏转录因子阴阳1号(YY1)的突变体上进行测试。VE或DE中YY1功能的丧失是致命的，导致肝芽和VE中HNF4的减少和血管内皮生长因子的丢失，以及两种组织中YY1-/-细胞邻近的间质中血管内皮生长因子诱导的血管生成的丧失。WT和YY1-/-VE和相关的卵黄囊间充质的转录图谱将被用来识别支持正常肝芽发育的依赖于血管内皮生长因子的旁分泌信号分子，以及调控肝芽中血管内皮生长因子的候选细胞自主信号分子。这些方法将被用来建立对肝芽发育的分子要求的更深层次的理解，并建立卵黄囊作为早期肝芽发育的遗传范例。这些进展不仅有助于更好地了解肝脏的正常发育和肝脏疾病的病理生物学基础，还将有助于开发从iPS或ES细胞生成功能性肝细胞的策略。

 DESCRIPTION (provided by applicant): The goal of this proposal is to test a novel system designed to elucidate the molecules support the emergence of the early liver bud from the definitive or embryonic endoderm (DE). We propose to gain such insights by using the yolk sac, which is not only functionally and structurally similar to the DE but also shares nearly identical transcriptional profile to the DE and almost all early liver bud markers are expressed in the yolk sac. Although a number of essential liver bud factors have been identified a gap in the field is an understanding of how loss of any one of these factors alters the complete transcriptional profile underlying liver budding. One issue that initially stymied the identification of gene function in liver budding is the requirement of many liver bud essential genes earlier in the visceral endoderm (VE) of the yolk sac. The yolk sac is the extraembryonic tissue that acts as a primitive gut; facilitating nutrient uptake, metabolic waste and gas exchange. Many early liver markers are expressed in the yolk sac and the conservation of genetic networks supports a hypothesis that during the evolution of mammals, the yolk sac co-opted the existing DE genetic program. Although inducible Cre strategies can now bypass genetic requirements in the VE, the difficulty in dissecting/isolating sufficient quantities of early liver bud tissues has prevented in depth genome-wide analyses on both normal and mutant tissue. To overcome these difficulties, we propose to use the yolk sac as a proxy for the early liver bud and to perform transcriptome analysis of mutant and WT yolk sacs to generate candidate factors similarly at play in the liver bud. This strategy will be tested on mutants lacking the transcription factor, Yin-yang 1 (YY1). Loss of YY1 function in either the VE or DE is lethal, resulting in reduced HNF4 and loss of VEGF in both the liver bud and VE as well as loss of VEGF induced angiogenesis in the mesenchyme adjacent to the YY1-/- cells in both tissues. Transcriptional profiling of WT and YY1-/- VE and associated yolk sac mesenchyme will be used to identify candidate VEGF-dependant paracrine signaling molecules that support normal liver bud development and to identify candidate cell-autonomous that regulate VEGF in the liver bud. These methods will be used to build a deeper understanding of the molecular requirements underlying liver bud development and to establish the yolk sac as a genetic paradigm for early liver bud development. Such advances will not only aid in better understanding normal liver development and pathobiology underlying liver disease, but will also aid in producing strategies that generate functional hepatocytes from iPS or ES cells.